Leukemia-initiating cells, also known as leukemic stem cells (LSCs), are experimentally defined by their ability to engraft immunocompromised mice and are believed to be a major cause of relapse in acute myeloid leukemia (AML). Despite the aggressive characteristics of acute leukemia, AML blasts are difficult to culture once removed from the patient, and LSCs, which are a minor fraction of the blast population, are especially difficult to transplant after culture. This impedes development of new therapies for AML that target LSCs. Here, we present a simple strategy to culture LSCs in cytokine-free medium and to perform flow cytometric analysis of the resulting cell population for the characterization of LSCs maintenance and differentiation.Acute myeloid leukemia (AML) is a highly frequent hematological malignancy, characterized by clinical and biological diversity, along with high relapse and mortality rates. The inherent functional and genetic intra-tumor heterogeneity in AML is thought to play an important role in disease recurrence and resistance to chemotherapy. Patient-derived xenograft (PDX) models preserve important features of the original tumor, allowing, at the same time, experimental manipulation and in vivo amplification of the human cells. Here we present a detailed protocol for the generation of fluorescently labeled AML PDX models to monitor cell proliferation kinetics in vivo, at the single-cell level. Although experimental protocols for cell proliferation studies are well established and widespread, they are not easily applicable to in vivo contexts, and the analysis of related time-series data is often complex to achieve. To overcome these limitations, model-driven approaches can be exploited to investigate different aspects of cell population dynamics. Among the existing approaches, the ProCell framework is able to perform detailed and accurate stochastic simulations of cell proliferation, relying on flow cytometry data. In particular, by providing an initial and a target fluorescence histogram, ProCell automatically assesses the validity of any user-defined scenario of intra-tumor heterogeneity, that is, it is able to infer the proportion of various cell subpopulations (including quiescent cells) and the division interval of proliferating cells. Here we explain the protocol in detail, providing a description of our methodology for the conditional expression of H2B-GFP in human AML xenografts, data processing by flow cytometry, and the final elaboration in ProCell.Intense chemotherapy regimens of patients diagnosed with T cell acute lymphoblastic leukemia (T-ALL) have proved successful for improving patient's overall survival, especially in children. But still T-ALL treatment remains challenging, since side effects of chemotherapeutic drugs often worsen patient's quality of life, and relapse rates remain significant. Hence, the availability of experimental animal models capable of recapitulating the biology of human T-ALL is obligatory as a critical tool to explore novel promising therapies directed against specific targets that have been previously validated in in vitro assays. For this purpose, patient-derived xenografts (PDX) of primary human T-ALL are currently of great interest as preclinical models for novel therapeutic strategies toward transition into clinical trials. In this chapter, we describe the lab workflow to perform PDX assays, from the initial processing of patient T-ALL samples, genetic in vitro modifications of leukemic cells by lentiviral transduction, inoculation routes, monitoring for disease development, and mouse organ examination, to administration of several treatments.Hematopoietic stem cells have the ability to produce all blood cells. When hematological malignancies occur, transplant of compatible blood or bone marrow cells from a healthy donor to the patient is an efficient solution to restore normal hematopoiesis. Bone marrow transplant in a mouse model is often used to study HSC function and capacity to repopulate an irradiated recipient. This protocol details the different steps of a competitive bone marrow transplant experiment, beginning with total body irradiation of the recipient mice; preparation and administration of the donor and competitor bone marrow samples; peripheral blood analysis to follow reconstitution posttransplant; and finally, the analysis of recipient bone marrow and secondary transplants to evaluate long-term HSC function. Different formulas used to establish transplant efficiency are explained. All the steps are discussed in detail, including tips, variations, and alternative procedures with their advantages and disadvantages.Hematopoietic stem cells (HSCs) display heterogeneity in their characteristic features of undergoing self-renewal and multipotency within the blood system. https://www.selleckchem.com/EGFR(HER).html While the same cell surface protein markers can be used to isolate HSCs from young and aged mice, recent studies have shown that their functional potential changes throughout the aging process. However, many of these conclusions have been the result of conventional HSC transplantation assays. These methods, though valuable, undermine not only the effective analysis of the underlying heterogeneity in aged HSC function, but also a full understanding of aged HSC differentiation potential to all five blood lineages. In this chapter, we describe a method to perform in vivo clonal analysis of aged HSCs using single-cell transplantation, incorporating a five-blood lineage tracing system using the Kusabira-Orange (KuO) trancsgenic mouse line.Leukemic stem cells are highly dynamic and heterogeneous. Analysis of leukemic stem cells at the single-cell level should provide a wealth of insights that would not be possible using bulk measurements. Mass spectrometry (MS)-based proteomic workflows can quantify hundreds or thousands of proteins from a biological sample and has proven invaluable for biomedical research, but samples comprising large numbers of cells are typically required due to limited sensitivity. Recent developments in sample processing, chromatographic separations, and MS instrumentation are now extending in-depth proteome profiling to single mammalian cells. Here, we describe specific techniques that increase the sensitivity of single-cell proteomics by orders of magnitude, enabling the promise of single-cell proteomics to become a reality. We anticipate such techniques can significantly advance the understanding of leukemic stem cells.